Elizabeth A. McCart

Enhanced hematopoietic protection from radiation by the combination of genistein and captopril.

The hematopoietic system is sensitive to radiation injury, and mortality can occur due to blood cell deficiency and stem cell loss. Genistein and the angiotensin converting enzyme (ACE) inhibitor captopril are two agents shown to protect the hematopoietic system from radiation injury. In this study we examined the combination of genistein with captopril for reduction of radiation-induced mortality from hematopoietic damage and the mechanisms of radiation protection. C57BL/6J mice were exposed to 8.25Gy (60)Co total body irradiation (TBI) to evaluate the effects of genistein and captopril alone and in combination on survival, blood cell recovery, hematopoietic progenitor cell recovery, DNA damage, and erythropoietin production. 8.25Gy TBI resulted in 0% survival after 30days in untreated mice. A single subcutaneous injection of genistein administered 24h before TBI resulted in 72% survival. Administration of captopril in the drinking water, from 1h through 30days postirradiation, increased survival to 55%. Genistein plus captopril increased survival to 95%. Enhanced survival was reflected in a reduction of radiation-induced anemia, improved recovery of nucleated bone marrow cells, splenocytes and circulating red blood cells. The drug combination enhanced early recovery of marrow progenitors: erythroid (CFU-E and BFU-E), and myeloid (CFU-GEMM, CFU-GM and CFU-M). Genistein alone and genistein plus captopril protected hematopoietic progenitor cells from radiation-induced micronuclei, while captopril had no effect. Captopril alone and genistein plus captopril, but not genistein alone, suppressed radiation-induced erythropoietin production. These data suggest that genistein and captopril protect the hematopoietic system from radiation injury via independent mechanisms.

Captopril modulates hypoxia-inducible factors and erythropoietin responses in a murine model of total body irradiation.

OBJECTIVE Our laboratory reported that the angiotensin converting enzyme inhibitor captopril improves erythroid recovery from total body irradiation (TBI) in mice when administered after irradiation. However, captopril administered before TBI attenuates erythroid recovery. Here we investigate captopril and radiation regulation of erythropoietin (EPO) and thrombopoietin (TPO), key effectors of erythroid progenitor proliferation and differentiation. MATERIALS AND METHODS C57BL/6 mice, nonirradiated or exposed to 7.5 Gy TBI ((60)Co, 0.6 Gy/min) were untreated or administered captopril. Plasma EPO and TPO levels were measured by enzyme-linked immunosorbent assay. Gene expression of EPO was determined by quantitative reverse transcription polymerase chain reaction. The hypoxia-inducible factors (HIF)-1α and -2α were measured by immunoblotting. RESULTS In nonirradiated mice, continuous captopril administration in the water transiently reduced reticulocytes and red blood cells after 7 and 10 days, respectively. EPO plasma levels and gene expression were reduced below detectable limits after 2 days of captopril treatment, but recovered within 7 days. HIF-1α and HIF-2α were activated preceding reticulocyte and red blood cell recovery. TBI, which ablates early and late-stage erythroid progenitors, activated both HIFs and increased EPO and TPO. Captopril treatment postirradiation suppressed radiation-induced HIF activation and EPO expression. In contrast, captopril administration for 7 days before TBI resulted in earlier EPO induction and activation. Captopril treatment lowered TPO levels in nonirradiated mice, but had minimal effects on radiation-induced TPO. CONCLUSIONS In nonirradiated mice, captopril biphasically regulates EPO via HIF activation. TBI ablates erythroid progenitors, resulting in hypoxia, HIF activation, and increased EPO expression that are modulated by captopril treatment. These data suggest that short-term suppression of radiation-induced EPO immediately after TBI is favorable for erythroid recovery.

Mechanism of Erythropoietin Regulation by Angiotensin II

Erythropoietin (EPO) is the primary regulator of red blood cell development. Although hypoxic regulation of EPO has been extensively studied, the mechanism(s) for basal regulation of EPO are not well understood. In vivo studies in healthy human volunteers and animal models indicated that angiotensin II (Ang II) and angiotensin converting enzyme inhibitors regulated blood EPO levels. In the current study, we found that Ang II induced EPO expression in situ in murine kidney slices and in 786-O kidney cells in culture as determined by reverse transcription polymerase chain reaction. We further investigated the signaling mechanism of Ang II regulation of EPO in 786-O cells. Pharmacological inhibitors of Ang II type 1 receptor (AT1R) and extracellular signal-regulated kinase 1/2 (ERK1/2) suppressed Ang II transcriptional activation of EPO. Inhibitors of AT2R or Src homology 2 domain–containing tyrosine phosphatase had no effect. Coimmunoprecipiation experiments demonstrated that p21Ras was constitutively bound to the AT1R; this association was increased by Ang II but was reduced by the AT1R inhibitor telmisartan. Transmembrane domain (TM) 2 of AT1R is important for G protein–dependent ERK1/2 activation, and mutant D74E in TM2 blocked Ang II activation of ERK1/2. Ang II signaling induced the nuclear translocation of the Egr-1 transcription factor, and overexpression of dominant-negative Egr-1 blocked EPO promoter activation by Ang II. These data identify a novel pathway for basal regulation of EPO via AT1R-mediated Egr-1 activation by p21Ras-mitogen-activated protein kinase/ERK kinase-ERK1/2. Our current data suggest that Ang II, in addition to regulating blood volume and pressure, may be a master regulator of erythropoiesis.

Effect of ionizing radiation on liver protein oxidation and metabolic function in C57BL/6J mice

Abstract Purpose: Protein oxidation in response to radiation results in DNA damage, endoplasmic reticulum stress/unfolded protein response, cell cycle arrest, cell death and senescence. The liver, a relatively radiosensitive organ, undergoes measurable alterations in metabolic functions following irradiation. Accordingly, we investigated radiation-induced changes in liver metabolism and alterations in protein oxidation. Materials and methods: C57BL/6 mice were sham irradiated or exposed to 8.5 Gy 60Co (0.6 Gy/min) total body irradiation. Metabolites and metabolic enzymes in the blood and liver tissue were analyzed. Two-dimensional gel electrophoresis and OxyBlot™ were used to detect carbonylated proteins that were then identified by peptide mass fingerprinting. Results: Analysis of serum metabolites revealed elevated glucose, bilirubin, lactate dehydrogenase (LDH), high-density lipoprotein, and aspartate aminotransferase within 24–72 h post irradiation. Liver tissue LDH and alkaline phosphatase activities were elevated 24–72 h post irradiation. OxyBlotting revealed that the hepatic proteome contains baseline protein carbonylation. Radiation exposure increased carbonylation of specific liver proteins including carbonic anhydrase 1, α-enolase, and regucalcin. Conclusions: 8.5 Gy irradiation resulted in distinct metabolic alterations in hepatic functions. Coincident with these changes, radiation induced the carbonylation of specific liver enzymes. The oxidation of liver enzymes may underlie some radiation-induced alterations in hepatic function.

Bone Marrow Protein Oxidation in Response to Ionizing Radiation in C57BL/6J Mice

The bone marrow is one of the most radio-sensitive tissues. Accidental ionizing radiation exposure can damage mature blood cells and hematopoietic progenitor/stem cells, and mortality can result from hematopoietic insufficiency and infection. Ionizing radiation induces alterations in gene and protein expression in hematopoietic tissue. Here we investigated radiation effects on protein carbonylation, a primary marker for protein oxidative damage. C57BL/6 mice were either sham irradiated or exposed to 7.5 Gy 60Co (0.6 Gy/min) total body irradiation. Bone marrow was obtained 24 h post-irradiation. Two dimensional (2-D) gel electrophoresis and Oxyblot immunodetection were used to discover carbonylated proteins, and peptide mass fingerprinting was performed for identification. 2D gels allowed the detection of 13 carbonylated proteins in the bone marrow; seven of these were identified, with two pairs of the same protein. Baseline levels of carbonylation were found in 78 kDa glucose-related protein, heat shock protein cognate 71 KDa, actin, chitinase-like protein 3 (CHI3L1), and carbonic anhydrase 2 (CAII). Radiation increased carbonylation in four proteins, including CHI3L1 and CAII, and induced carbonylation of one additional protein (not identified). Our findings indicate that the profile of specific protein carbonylation in bone marrow is substantially altered by ionizing radiation. Accordingly, protein oxidation may be a mechanism for reduced cell viability.

Tsc2 disruption in mesenchymal progenitors results in tumors with vascular anomalies overexpressing Lgals3

Increased mTORC1 signaling from TSC1/TSC2 inactivation is found in cancer and causes tuberous sclerosis complex (TSC). The role of mesenchymal-derived cells in TSC tumorigenesis was investigated through disruption of Tsc2 in craniofacial and limb bud mesenchymal progenitors. Tsc2cKOPrrx1-cre mice had shortened lifespans and extensive hamartomas containing abnormal tortuous, dilated vessels prominent in the forelimbs. Abnormalities were blocked by the mTORC1 inhibitor sirolimus. A Tsc2/mTORC1 expression signature identified in Tsc2-deficient fibroblasts was also increased in bladder cancers with TSC1/TSC2 mutations in the TCGA database. Signature component Lgals3 encoding galectin-3 was increased in Tsc2-deficient cells and serum of Tsc2cKOPrrx1-cre mice. Galectin-3 was increased in TSC-related skin tumors, angiomyolipomas, and lymphangioleiomyomatosis with serum levels in patients with lymphangioleiomyomatosis correlating with impaired lung function and angiomyolipoma presence. Our results demonstrate Tsc2-deficient mesenchymal progenitors cause aberrant morphogenic signals, and identify an expression signature including Lgals3 relevant for human disease of TSC1/TSC2 inactivation and mTORC1 hyperactivity. DOI: http://dx.doi.org/10.7554/eLife.23202.001

Hepatocyte Growth Factor Isoforms in Tissue Repair, Cancer, and Fibrotic Remodeling

Hepatocyte growth factor (HGF), also known as scatter factor (SF), is a pleotropic factor required for normal organ development during embryogenesis. In the adult, basal expression of HGF maintains tissue homeostasis and is up-regulated in response to tissue injury. HGF expression is necessary for the proliferation, migration, and survival of epithelial and endothelial cells involved in tissue repair in a variety of organs, including heart, lung, kidney, liver, brain, and skin. The administration of full length HGF, either as a protein or using exogenous expression methodologies, increases tissue repair in animal models of tissue injury and increases angiogenesis. Full length HGF is comprised of an N-terminal hairpin turn, four kringle domains, and a serine protease-like domain. Several naturally occurring alternatively spliced isoforms of HGF were also identified. The NK1 variant contains the N-terminal hairpin and the first kringle domain, and the NK2 variant extends through the second kringle domain. These alternatively spliced forms of HGF activate the same receptor, MET, but they differ from the full length protein in their cellular activities and their biological functions. Here, we review the species-specific expression of the HGF isoforms, their regulation, the signal transduction pathways they activate, and their biological activities.

Protein Oxidation in the Lungs of C57BL/6J Mice Following X-Irradiation

Damage to normal lung tissue is a limiting factor when ionizing radiation is used in clinical applications. In addition, radiation pneumonitis and fibrosis are a major cause of mortality following accidental radiation exposure in humans. Although clinical symptoms may not develop for months after radiation exposure, immediate events induced by radiation are believed to generate molecular and cellular cascades that proceed during a clinical latent period. Oxidative damage to DNA is considered a primary cause of radiation injury to cells. DNA can be repaired by highly efficient mechanisms while repair of oxidized proteins is limited. Oxidized proteins are often destined for degradation. We examined protein oxidation following 17 Gy (0.6 Gy/min) thoracic X-irradiation in C57BL/6J mice. Seventeen Gy thoracic irradiation resulted in 100% mortality of mice within 127–189 days postirradiation. Necropsy findings indicated that pneumonitis and pulmonary fibrosis were the leading cause of mortality. We investigated the oxidation of lung proteins at 24 h postirradiation following 17 Gy thoracic irradiation using 2-D gel electrophoresis and OxyBlot for the detection of protein carbonylation. Seven carbonylated proteins were identified using mass spectrometry: serum albumin, selenium binding protein-1, alpha antitrypsin, cytoplasmic actin-1, carbonic anhydrase-2, peroxiredoxin-6, and apolipoprotein A1. The carbonylation status of carbonic anhydrase-2, selenium binding protein, and peroxiredoxin-6 was higher in control lung tissue. Apolipoprotein A1 and serum albumin carbonylation were increased following X-irradiation, as confirmed by OxyBlot immunoprecipitation and Western blotting. Our findings indicate that the profile of specific protein oxidation in the lung is altered following radiation exposure.

Captopril mitigates splenomegaly and myelofibrosis in the Gata1 low murine model of myelofibrosis

Allogeneic stem cell transplantation is currently the only curative therapy for primary myelofibrosis (MF), while the JAK2 inhibitor, ruxolitinib. Has been approved only for palliation. Other therapies are desperately needed to reverse life‐threatening MF. However, the cell(s) and cytokine(s) that promote MF remain unclear. Several reports have demonstrated that captopril, an inhibitor of angiotensin‐converting enzyme that blocks the production of angiotensin II (Ang II), mitigates fibrosis in heart, lung, skin and kidney. Here, we show that captopril can mitigate the development of MF in the Gata1low mouse model of primary MF. Gata1low mice were treated with 79 mg/kg/d captopril in the drinking water from 10 to 12 months of age. At 13 months of age, bone marrows were examined for fibrosis, megakaryocytosis and collagen expression; spleens were examined for megakaryocytosis, splenomegaly and collagen expression. Treatment of Gata1low mice with captopril in the drinking water was associated with normalization of the bone marrow cellularity; reduced reticulin fibres, splenomegaly and megakaryocytosis; and decreased collagen expression. Our findings suggest that treating with the ACE inhibitors captopril has a significant benefit in overcoming pathological changes associated with MF.

Abstract 1576: Mesenchymal disruption of Tsc2 in mice results in highly vascular hamartomas.

Patients with tuberous sclerosis complex (TSC) suffer from the formation of multi-organbenign tumors. Skin tumors from TSC patients are characterized by increased vascularity, fibrosis, and overactivation of mTORC1 signaling due to inactivation of either of the tumor suppressor genes TSC1 or TSC2 in fibroblasts. The role of the TSC2 gene in cells of mesenchymal origin with respect to tumor formation still remains unclear. To investigate the roles of TSC2 in these cells, we used the cre/loxP system to conditionally disrupt mouse Tsc2 in tissues derived from mesenchymal cells. Mice with floxed Tsc2 alleles were mated with mice expressing the cre recombinase transgene under the control of a Prrx1 regulatory element (Prrx1-cre), which is selectively expressed in craniofacial and limb bud mesenchyme. Cells with homozygous Tsc2 floxed alleles expressing the Prrx1-cre transgene (termed here “Tsc2cKO mice”) efficiently eliminated Tsc2 expression from fibroblasts isolated from limb and ventral skin. An EYFP reporter transgene was also present to track cells expressing cre recombinase. Fibroblasts grown from the skin of Tsc2cKO mice expressed EYFP, lacked Tsc2 protein and had elevated levels of phosphorylated ribosomal protein S6, a marker of mTORC1 signaling. Tsc2cKO mice had shortened lifespan with a median survival between 5 and 6 months of age. Tumors were observed in spleen and forepaws of nearly all mice, and tumors involving the liver, skin, skeletal muscle, mediastinum, and kidney also occurred. These results were unexpected due to the lineage restriction of the Prrx1-cre transgene. Tumor formation began to appear on forepaws near the heel of the paw pad as early as 3 weeks of age. By 6 weeks of age the lesions generally showed a reddish appearance. In mice serially imaged by MRI, splenic tumors were apparent by 10-12 weeks of age and renal tumors were apparent by 7-8 weeks. Histologically, the forepaw, splenic, and hepatic tumors were benign fibrovascular proliferations with perivascular cells that stained with smooth muscle actin, most prominently in the spleen. Using ex vivo fluorescent imaging, EYFP fluorescence was not detected in internal organs of control mice containing Prrx1-cre, but strong fluorescence was observed in the splenic, hepatic, and renal tumors of Tsc2cKO mice. In summary, we have developed model system to study the role of Tsc2-deficient mesenchymal cells in hamartoma formation. Citation Format: Peter Klover, Rajesh Thangapazham, Shaowei Li, Jiro Kato, Stasia A. Anderson, Victoria Hoffman, Ji-an Wang, Joshua Bernstock, Elizabeth McCart, Joel Moss, Thomas Darling. Mesenchymal disruption of Tsc2 in mice results in highly vascular hamartomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1576. doi:10.1158/1538-7445.AM2013-1576